Heterodimeric follicle stimulating hormone-Fc (FSH-Fc) fusion proteins for the treatment of infertility

ABSTRACT

The invention provides novel heterodimeric fusion proteins comprising a first polypeptide including an alpha subunit of FSH (αFSH) linked directly or indirectly to a binding partner of neonatal Fc receptor (FcRn) and a second polypeptide including a beta subunit of FSH (βFSH) linked directly or indirectly to an FcRn binding partner. In one embodiment the FcRn binding partner includes an Fc fragment of an immunoglobulin, e.g., an Fc fragment of IgG. Also provided are methods making and using the fusion proteins of the invention. The invention provides a method for increasing fertility in a subject and a method for treating a subject having a disease state responsive to treatment by FSH.

RELATED APPLICATION

This application claims benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application Ser. No. 60/540,236, filed on Jan. 28,2004, the entire content of which is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to the field of therapeutics forreproductive disorders. More specifically, the invention relates toheterodimeric follicle stimulating hormone-Fc fusion proteins for thetreatment of infertility.

BACKGROUND OF THE INVENTION

Infertility affects one in ten couples, resulting in millions of couplesstruggling to become pregnant. Many of these couples are potentialcandidates for infertility treatment. Follicle stimulating hormone(FSH), either extracted from urine or produced recombinantly, is aparenterally-administered protein product used by specialists toincrease fertility and has been so used clinically since the 1960's. Forexample, FSH is used for ovulation induction (OI) and for controlledovarian hyperstimulation (COH). Whereas OI is directed at achieving asingle follicle to ovulate, COH is directed at harvesting multipleoocytes for use in various in vitro assisted reproductive technologies(e.g., for in vitro fertilization). FSH is also used in gonadotropinreplacement therapy in males.

The use of FSH is limited by its high cost, the need for extensivemonitoring by specialist physicians, by lack of oral dosing or othernoninvasive routes of administration, and the need for daily patientinjections. Recombinant FSH suffers from a short half-life andcorrespondingly diminished biopotency, necessitating frequentadministration and limited clinical usefulness. For example, recombinanthuman FSH (hFSH) must be administered as a daily intramuscular orsubcutaneous injection, often for 8 to 12 days or more when used forovulation induction. These regimens are associated with a number of sideeffects, including local irritation and discomfort, which result in poorcompliance and a reduction in therapeutic efficacy. Thus, there exists aneed for forms of FSH with increased half-life and bioavailability ascompared to traditional forms of FSH therapy.

Follicle stimulating hormone (FSH) is found in nature as anon-covalently linked heterodimeric protein consisting of an alpha (α)subunit and a beta (β) subunit (Pierce J G and Parsons T F (1981) AnnRev Biochem 50:465-95). Subunit assembly has been reported to beessential for bioactivity of FSH (Jia X C and Hseuh A J W (1986)Endocrinology 119:1570-7) as well as for the stability of the betasubunit (Keene et al. (1989) J Biol Chem 264:4769-75).

One approach to improve FSH therapy has been by increasing theglycosylation of the protein. Other approaches have included carboxyterminal portion (CTP) extended forms of FSH (see e.g., U.S. Pat. No.5,338,835, and U.S. 2003/0211580 A1) or FSH mimetics (see e.g., U.S.Pat. No. 6,653,338) for the treatment of infertility. Early attempts,however, to improve half-life and bioactivity have yet to result in atherapeutically effective drug capable of providing advantages overexisting therapies.

It has also been reported that a single-chain fusion of the α and βsubunits of FSH (single-chain FSH) is fully active (Sugahara et al.(1996) J Biol Chem 271:10445-8). Single-chain FSH is reported to have anincreased serum half-life when fused with the carboxyterminal peptide ofhuman chorionic gonadotropin (hCG). (See Klein et al. (2003) Hum Reprod18:50-6; Bouloux et al. (2001) Hum Reprod 16:1592-7; Duijkers et al.(2002) Hum Reprod 17:1987-93.)

The use of heterodimeric FSH and its formulation are fraught withstability and purification issues not present with single-chain FSH.While recombinant FSH is known (see e.g., U.S. Pat. No. 5,767,251),keeping the alpha and beta subunits associated in a way that results ina biologically active molecule useful for therapeutic purposes, and inparticular with long-acting forms of heterodimeric FSH, remained achallenge until the present invention.

The creation of fusion proteins comprised of immunoglobulin constantregions linked to a protein of interest, or fragment thereof, has beendescribed (see, e.g., U.S. Pat. Nos. 5,155,027, 5,428,130, 5,480,981,and 5,808,029). These molecules usually possess both the biologicalactivity associated with the linked molecule of interest as well as theeffector function, or some other desired characteristic, associated withthe immunoglobulin constant region. Fusion proteins comprising an Fcportion of an immunoglobulin can bestow several desirable properties ona fusion protein including increased stability, increased serumhalf-life (see Capon et al. (1989) Nature 337:525) as well as binding toFc receptors such as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos.6,086,875, 6,030,613, and 6,485,726).

FcRn is active in adult epithelial tissue and expressed in the lumen ofthe intestines, pulmonary airways, nasal surfaces, vaginal surfaces,colon and rectal surfaces (U.S. Pat. No. 6,485,726). Fusion proteinscomprised of FcRn binding partners (e.g., IgG, Fc fragments) can beeffectively shuttled across epithelial barriers by FcRn, thus providinga non invasive means to systemically administer a desired therapeuticmolecule. Additionally, fusion proteins comprising an FcRn bindingpartner are endocytosed and protected by cells expressing the FcRn.Instead of being marked for degradation, these fusion proteins arerecycled out into circulation again, thus increasing the in vivohalf-life of these proteins.

FSH has been conjugated to Fc as described in U.S. 2003/0235536 A1.However, therein is described single-chain FSH-Fc fusion protein fordelivery to the central airways of non-human primates. Single-chainFSH-Fc fusions contain hFSHβα-Fc, in a single-chain with the beta andalpha subunits conjugated end-to-end and the two polypeptide chains ofthe fusion are identical (see FIG. 1 c). As shown in FIG. 1 c,single-chain FSH-Fc forms a homodimer.

SUMMARY OF THE INVENTION

The invention relates to fusion proteins of heterodimeric FSH whereinthe alpha and beta subunits of FSH are each conjugated to an FcRnbinding partner or to an Fc fragment. In one embodiment, the inventionprovides fusion proteins having two polypeptide chains, one chain havingat least αFSH, linked directly or indirectly through an optional linkerto an Fc fragment of an immunoglobulin, and the second chain havingβFSH, also linked directly or indirectly through an optional linker toan Fc fragment of an immunoglobulin. By way of these fusion proteins,the invention provides methods for increasing the half-life of FSH and,therefore, further provides an effective means for increasing asubject's fertility with reduced dosing frequency and/or treating adisease state responsive to FSH therapy.

In contrast to the single-chain FSH-Fc fusion protein described in U.S.2003/0235536 A1, the heterodimeric FSH-Fc fusion proteins of the presentinvention (FIG. 1 c) have the alpha subunit of FSH conjugated to one Fcchain and the beta subunit of FSH conjugated to the other Fc chain,wherein the alpha and beta subunits are aligned head-to-head andtail-to-tail and the Fc fragments are similarly aligned head-to-head andtail-to-tail. The two chains are brought into association with oneanother by the interactions between the alpha and beta subunits of FSHas exist, for example, with endogenous FSH. Alternatively, or inaddition thereto, the association of the two Fc chains, as for examplethrough a disulfide bond(s), brings the alpha and beta subunits of FSHin proximity to each other thus enhancing its bioactivity as compared tosingle-chain FSH fusion proteins.

In one aspect the invention is a heterodimeric fusion protein includingtwo associated polypeptide chains, the first chain including an alphasubunit of follicle stimulating hormone (αFSH) conjugated to a neonatalFc receptor (FcRn) binding partner and the second chain including a betasubunit of FSH (βFSH) conjugated to an FcRn binding partner, wherein thehead of αFSH is aligned with the head of βFSH and the tails of each ofthe respective FcRn binding partners are aligned.

In one aspect the invention is a fusion protein including twopolypeptide chains of the formulaαFSH-L-Fc:βFSH-L-Fcwherein αFSH is an alpha subunit of FSH, βFSH is a beta subunit of FSH,L is a linker or direct bond, and Fc is an Fc fragment of animmunoglobulin, wherein carboxy termini of αFSH and βFSH are linkedeither directly or indirectly through L to the amino terminus of therespective Fc, further wherein the colon (:) represents an associationbetween the two polypeptide chains of the fusion protein, and furtherwherein the head of αFSH is aligned with the head of βFSH and the tailsof each of the respective Fc fragments are aligned.

In one aspect the invention is a fusion protein including twopolypeptide chains of the formulaFc-L-αFSH: Fc-L-βFSHwherein αFSH is the alpha subunit of FSH, βFSH is the beta subunit ofFSH, L is a linker or direct bond, and Fc is an Fc fragment of animmunoglobulin, wherein the amino termini of αFSH and βFSH are linkedeither directly or indirectly through L to the carboxy terminus of therespective Fc, further wherein the colon (:) represents an associationbetween the two polypeptide chains of the fusion protein, and furtherwherein the head of αFSH is aligned with the head of βFSH and the tailsof each of the respective Fc fragments are aligned.

In one embodiment the fusion protein of the invention can include atleast one tag moiety. The tag moiety can be used, for example, to assistin purification or identification of recombinantly produced polypeptideor protein. For example, in one embodiment one polypeptide of theheterodimeric fusion protein further includes a histidine tag.

The invention in one aspect is a pharmaceutical composition whichincludes a fusion protein of the invention and a pharmaceuticallyacceptable excipient.

In another aspect the invention is a method for increasing a subject'sfertility. The method according to this aspect of the invention includesthe step of administering to the subject an amount of a fusion proteinof the invention effective to enhance fertility of the subject.

In one aspect the invention is a method for treating a subject having adisease state responsive to treatment by FSH. The method according tothis aspect of the invention includes the step of administering to thesubject an effective amount of a fusion protein of the invention.

In one aspect the invention is a method of increasing the half-life ofheterodimeric FSH. The method according to this aspect of the inventionincludes the step of conjugating, directly or indirectly through alinker, each of an alpha subunit and a beta subunit of FSH to an FcRnbinding partner, wherein the head of the αFSH is aligned with the headof βFSH and the tails of each of the respective FcRn binding partnersare aligned.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of schematic diagrams depicting (a) DNA constructmade to create single-chain FSH-Fc protein, (b) DNA constructs made tocreate heterodimer FSH-Fc protein, and (c) single-chain FSH-Fc andheterodimer FSH-Fc proteins.

FIG. 2 is an image of an SDS-PAGE gel of single-chain FSH-Fc andheterodimer FSH-Fc run under reducing and non-reducing conditions. Lane1, single-chain FSH-Fc reduced; lane 2, heterodimer FSH-Fc reduced; lane3, single-chain FSH-Fc non-reduced; and lane 4, heterodimer FSH-Fcnon-reduced.

FIG. 3 is a bar graph depicting ovarian weight in 21-day-old female ratstreated with a single subcutaneous dose of 1 nmol/kg recombinant FSH,single-chain FSH-Fc, or heterodimer FSH-Fc. Ovarian weight was measured72 h after dosing. Data are presented as average ovarian weight±standarddeviation (SD). n=10/group.

FIG. 4 is a graph depicting levels of single-chain FSH-Fc (circles) andheterodimer FSH-Fc (triangles) in neonatal rat serum after a single oraldose of 0.3 mg/kg. n=4/timepoint.

FIG. 5 is an image of an SDS-PAGE gel showing the effects of excess IgGon the oral uptake of single-chain FSH-Fc and heterodimer FSH-Fc inneonatal rats. Lane 1, 50,000 cpm input single-chain FSH-Fc; Lane 2,¹²⁵I-single-chain FSH-Fc; Lane 3, ¹²⁵I-single-chain FSH-Fc in thepresence of excess IgG; Lane 4, 50,000 cpm input heterodimer FSH-Fc;Lane 5, ¹²⁵I-heterodimer FSH-Fc; Lane 6, ¹²⁵I-heterodimer FSH-Fc in thepresence of excess IgG.

FIG. 6 is a pair of bar graphs depicting testis weight in two-day-oldmale rats treated daily for 14 days with single oral doses (1 nmol/kg)of (a) recombinant FSH or single-chain FSH-Fc, and (b) single-chainFSH-Fc or heterodimer FSH-Fc. Data presented as average testisweight±SD. n=6-10/group.

FIG. 7 is a pair of graphs depicting concentration-time profiles of (a)single-chain FSH-Fc and (b) heterodimer FSH-Fc in cynomolgus monkeyserum following a single deposited pulmonary dose of 45 μg/kg. Eachcurve is representative of a single monkey.

FIG. 8 is a pair of graphs depicting inhibin B concentration-timeprofiles in cynomolgus monkey serum following a single depositedpulmonary dose of 45 μg/kg of (a) single-chain FSH-Fc and (b)heterodimer FSH-Fc. Each curve is representative of a single monkey.

DETAILED DESCRIPTION DEFINITIONS

Affinity tag, as used herein, means a molecule attached to a secondmolecule of interest, capable of interacting with a specific bindingpartner for the purpose of isolating or identifying said second moleculeof interest.

Analogs of, or proteins or peptides or substantially identical to thefusion proteins of the invention, as used herein, means that a relevantamino acid sequence of a protein or a peptide is at least 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a given sequence. Byway of example, such sequences may be variants derived from variousspecies, or they may be derived from the given sequence by truncation,deletion, amino acid substitution or addition. Percent identity betweentwo amino acid sequences is determined by standard alignment algorithmssuch as, for example, Basic Local Alignment Tool (BLAST) described inAltschul et al. (1990) J. Mol. Biol., 215:403-410; the algorithm ofNeedleman et al. (1970) J. Mol. Biol., 48:444-453; the algorithm ofMeyers et al. (1988) Comput Appl. Biosci., 4:11-17; or Tatusova et al.(1999) FEMS Microbiol. Lett., 174:247-250, etc. Such algorithms areincorporated into the BLASTN, BLASTP and “BLAST 2 Sequences” programs.When utilizing such programs, the default parameters can be used. Forexample, for nucleotide sequences the following settings can be used for“BLAST 2 Sequences”: program BLASTN, reward for match 2, penalty formismatch −2, open gap and extension gap penalties 5 and 2 respectively,gap x_dropoff 50, expect 10, word size 11, filter ON. For amino acidsequences the following settings can be used for “BLAST 2 Sequences”:program BLASTP, matrix BLOSUM62, open gap and extension gap penalties 11and 1 respectively, gap x_dropoff 50, expect 10, word size 3, filter ON.

Bioavailability, as used herein, means the extent and/or rate at which asubstance is absorbed into a living system or is made available at thesite of physiological activity.

DNA construct, as used herein, means a DNA molecule, or a clone of sucha molecule, either single- or double-stranded, that has been modifiedthrough human intervention to contain segments of DNA combined in amanner that as a whole would not otherwise exist in nature. DNAconstructs contain the information necessary to direct the expression ofpolypeptides of interest. DNA constructs can include promoters,enhancers and transcription terminators. DNA constructs containing theinformation necessary to direct the secretion of a polypeptide willtypically also contain at least one secretory signal sequence.

A fragment, as used herein with respect to a peptide, polypeptide, orprotein, refers to a peptide or polypeptide comprising an amino acidsequence of at least 2 contiguous amino acid residues, of at least 5contiguous amino acid residues, of at least 10 contiguous amino acidresidues, of at least 15 contiguous amino acid residues, of at least 20contiguous amino acid residues, of at least 25 contiguous amino acidresidues, of at least 40 contiguous amino acid residues, of at least 50contiguous amino acid residues, of at least 100 contiguous amino acidresidues, or of at least 200 contiguous amino acid residues or anydeletion or truncation of a protein.

FSH and, equivalently except where otherwise noted, heterodimeric FSH,refer to a heterodimeric follicle stimulating hormone glycoproteincomposed of an alpha subunit (αFSH; FSH alpha) and a beta subunit (βFSH;FSH beta). FSH and heterodimeric FSH shall refer to naturally occurringforms of FSH and recombinant analogs thereof. In humans the alpha andbeta subunits are encoded in separate genes on separate chromosomes. FSHand heterodimeric FSH are to be distinguished from single-chain FSH andsingle-chain FSH fusion proteins, as well as from the heterodimeric FSHfusion proteins of the instant invention.

A fusion protein, as used herein, refers to any protein comprised of afirst amino acid sequence derived from a first source, bonded,covalently or non-covalently, to a second amino acid sequence derivedfrom a second source, wherein the first and second source are not thesame. A first source and a second source that are not the same caninclude two different biological entities, or two different proteinsfrom the same biological entity, or a biological entity and anon-biological entity. A fusion protein can include for example, aprotein derived from at least two different biological sources. Abiological source can include any non-synthetically produced nucleicacid or amino acid sequence (e.g., a genomic or cDNA sequence, an RNAsequence, a plasmid or viral vector, a native virion or a mutant oranalog, as further described herein, of any of the above). A syntheticsource can include a protein or nucleic acid sequence producedchemically and not by a biological system (e.g., solid phase synthesisof amino acid sequences). A fusion protein can also include a proteinderived from at least 2 different synthetic sources or a protein derivedfrom at least one biological source and at least one synthetic source.

Linked, as used herein with respect to nucleic acid sequences, refers toa first nucleic acid sequence covalently joined to a second nucleic acidsequence. The first nucleic acid sequence can be directly joined orjuxtaposed to the second nucleic acid sequence or alternatively anintervening sequence or linker moiety can covalently join the firstsequence to the second sequence. Linked, as used herein with respect toamino acid sequences, refers to a first amino acid sequence covalentlyjoined to a second amino acid sequence. The first amino acid sequencecan be directly joined or juxtaposed to the second amino acid sequenceor alternatively an intervening sequence or linker moiety can covalentlyjoin the first amino acid sequence to the second amino acid sequence.Linked can also refer to a first amino acid sequence non-covalentlyjoined to a second amino acid sequence.

Moderate stringency, as used herein with respect to nucleic acidhybridization, includes conditions that can be readily determined bythose having ordinary skill in the art based on, for example, the lengthof the DNA. The basic conditions are set forth by Sambrook et al.Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104,Cold Spring Harbor Laboratory Press (1989), and include use of aprewashing solution for the nitrocellulose filters 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0), hybridization conditions of 50% formamide, 6×SSC at42° C. (or other similar hybridization solution, such as Stark'ssolution, in 50% formamide at 42° C.), and washing conditions of 60° C.,0.5×SSC, 0.1% SDS.

High stringency, as used herein with respect to nucleic acidhybridization, includes conditions readily determined by the skilledartisan based on, for example, the length of the DNA. Generally, suchconditions are defined as hybridization conditions as above, and withwashing at approximately 68° C., 0.2×SSC, 0.1% SDS. The skilled artisanwill recognize that the temperature and wash solution salt concentrationcan be adjusted as necessary according to factors such as the length ofthe probe.

Polypeptide, as used herein, refers to a polymer of amino acids and doesnot refer to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term does not exclude post-expression modifications ofthe polypeptide, for example, glycosylation, acetylation,phosphorylation, pegylation, addition of a lipid moiety, or the additionof any organic or inorganic molecule. Included within the definitionare, for example, polypeptides containing one or more analogs of anamino acid (including, for example, unnatural amino acids) andpolypeptides with substituted linkages, as well as other modificationsknown in the art, both naturally occurring and non-naturally occurring.

Serum (or plasma) half-life, abbreviated “t_(1/2)”, as used herein meanselimination half-life, i.e., the time taken for the serum (or plasma)concentration of an agent to fall by one half. The related term“increased serum half-life” is used herein to reference a heterodimericFSH-Fc fusion protein that is cleared at a slower rate than native FSHalone (whether endogenous, recombinant or synthetic forms thereof.

Treat, treatment, and treating, as used herein, mean any of thefollowing: reduction in severity of any disorder or disease stateresponsive to treatment by therapy; the prophylaxis of one or moresymptoms associated with such disorders or disease states; the reductionin the duration of a disease course of such abnormalities; the provisionof beneficial effects to a subject having such abnormalities, withoutnecessarily curing an abnormality or disorder. As used herein withrespect to FSH, treat, treatment, and treating mean any of thefollowing: reduction in severity of any reproductive disorder or diseasestate responsive to treatment by FSH therapy; the prophylaxis of one ormore symptoms associated with such disorders or disease states; thereduction in the duration of a disease course of FSH abnormalities; theprovision of beneficial effects to a subject having such abnormalities(e.g., increasing fertility), without necessarily curing an FSHabnormality or reproductive disorder.

A. Improved Therapeutics for Infertility

The invention relates generally to improved therapeutics forreproductive disorders or disease states associated with thereproductive system, and in particular, FSH abnormalities. The inventionthus relates to a fusion protein of heterodimeric FSH wherein the alphaand beta subunits of FSH are each respectively conjugated to an Fcfragment or to an FcRn binding partner. The fusion proteins of theinvention have increased stability and improved half-life as compared toknown therapeutic agents used in reproductive, and/or FSH, therapy. Thefusion proteins of the invention can be administered parenterally ornon-invasively. While current FSH therapeutics are generallyadministered by subcutaneous or intramuscular injections, the fusionproteins of the invention can be administered by less invasive means,such as oral administration, nasal administration, or pulmonaryadministration. Current therapy requires daily injections, whereas thepresent invention may provide for less frequent parenteral, oral, orpulmonary dosing.

B. Fusion Proteins

The invention relates to fusion proteins of heterodimeric FSH whereinthe alpha and beta subunits of FSH are each conjugated to an FcRnbinding partner or to an Fc fragment. More specifically, the inventionin one embodiment provides fusion proteins having two polypeptidechains, one chain having at least αFSH, linked directly or indirectlythrough an optional linker, to an FcRn binding partner, and the secondchain having βFSH, also linked directly or indirectly through anoptional linker to an FcRn binding partner. In the subject fusions, thehead of αFSH is aligned with the head of βFSH and the tails of therespective FcRn binding partners are aligned. In one embodiment of theinvention, there is a linker between the FSH subunits and the respectiveFcRn binding partners.

In another embodiment of the invention, the fusion protein comprises twopolypeptide chains of the formulaαFSH-L-Fc:βFSH-L-Fcwherein αFSH is the alpha subunit of FSH, βFSH is the beta subunit ofFSH, L is a linker or direct bond, Fc is an Fc fragment of animmunoglobulin, and the colon (:) represents an association between thetwo polypeptide chains of the fusion. In this embodiment, the carboxytermini of αFSH and βFSH are linked either directly or indirectlythrough L to the amino terminus of the respective Fc and the head ofαFSH is aligned with the head of βFSH and the tails of each of therespective Fc fragments are aligned.

In an alternative embodiment, the fusion protein comprises twopolypeptide chains of the formulaFc-L-αFSH:Fc-L-βFSHwherein all aspects of the fusion are as described above for thepreceding embodiment, excepting that the amino termini of αFSH and βFSHare linked either directly or indirectly through L to the carboxyterminus of the respective Fc.

The association between the two polypeptide chains can be the result ofan association between the alpha and beta subunits of FSH and/or theresult of an association between the Fc fragments or FcRn bindingpartners. For example, in the instance of an association of the alphaand beta subunits of FSH, the interaction may be a non-covalentinteraction, e.g., an ionic interaction, a hydrophobic or hydrophilicinteraction, a Van der Waals interaction, and/or a hydrogen bond, e.g.,leucine zipper. Non-covalent association or interaction can involveinterdigitation of amphiphilic peptides such as, but not limited to,alpha helices, charge-charge interactions of amino acids bearingopposite charges, such as, but not limited to, lysine and aspartic acid,arginine and glutamic acid. In one embodiment the non-covalentassociation or interaction involves a leucine zipper comprising apeptide having several repeating amino acids in which every seventhamino acid is a leucine residue. (See, e.g., Branden et al. (1991)Introduction To Protein Structure, Garland Publishing, New York). In thecase of interactions between the Fc fragments, generally, they will becovalent bonds, and are generally one or two disulfide bonds as is foundin many of the Fc fragments of immunoglobulins. Therefore, in certainembodiments of the invention, there exists at least one disulfide bridgebetween the Fc fragments of the subject fusion.

In certain embodiments of the invention, conjugated to the subjectfusion, or one polypeptide chain thereof, can be a second linker oralternatively a tag that can be used to facilitate purification of thefusion protein, e.g., a FLAG tag, a histidine tag (see Example 1), a GSTtag, a maltose binding protein tag, or others known in the art.

1. Fusion Protein Variants

Derivatives and analogs of the fusion proteins of the invention,antibodies against the fusion proteins of the invention, and antibodiesagainst binding partners of the fusion proteins of the invention are allcontemplated, and can be made by altering their amino acid sequences bysubstitutions, additions, and/or deletions/truncations or by introducingchemical modifications that result in functionally equivalent molecules.It will be understood by one of ordinary skill in the art that certainamino acids in a sequence of any protein may be substituted for otheramino acids without adversely affecting the activity of the protein.

Various changes may be made in the amino acid sequences of the fusionproteins of the invention or DNA sequences encoding therefor withoutappreciable loss of their biological activity, function, or utility.Derivatives, analogs, or mutants resulting from such changes and the useof such derivatives is within the scope of the present invention. In aspecific embodiment, the derivative is functionally active, i.e.,capable of exhibiting one or more activities associated with the fusionproteins of the invention, e.g., increased fertility, increased eggproduction, increased spermatogenesis.

Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs (see Table1). Furthermore, various amino acids are commonly substituted withneutral amino acids, e.g., alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine (see, e.g., MacLennanet al. (1998) Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al.(1998) Adv. Biophys. 35:1-24). TABLE 1 Amino Acids and TheirSubstitutions Original Exemplary Typical Residues SubstitutionsSubstitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn(N) Gln Gln Asp (D) Glu Glu Cys (C) Ser, Ala Ser Gln (Q) Asn Asn Gly (G)Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala,Phe, Leu Norleucine Leu (L) Norleucine, Ile, Val, Met, Ile Ala, Phe Lys(K) Arg, 1,4-Diamino-butyric Arg Acid, Gln, Asn Met (M) Leu, Phe, IleLeu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Gly Ser (S) Thr,Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr,Ser Phe Val (V) Ile, Met, Leu, Phe, Ala, Leu Norleucine2. Heterodimeric Follicle Stimulating Hormone

The fusion proteins of the instant invention include heterodimeric FSH(alternatively referred to as “FSH heterodimer”). Recombinantheterodimeric FSH is described in U.S. Pat. No. 5,767,251. In anembodiment of the invention the FSH is human FSH (hFSH). Exemplarylibraries containing the beta and/or alpha subunits of FSH are describedin the ensuing Examples, and nucleotide sequences for alpha and betasubunits of human FSH are publicly available through GenBank asaccession numbers NM_(—)000735 and NM_(—)000510, respectively.

3. Immunoglobulins

Immunoglobulins are comprised of four protein chains that associatecovalently—two heavy chains and two light chains. Each chain is furthercomprised of one variable region and one constant region. Depending uponthe immunoglobulin isotype (i.e., IgG, IgM, IgA, IgD, IgE), the heavychain constant region is comprised of 3 or 4 constant region domains(e.g., CH1, CH2, CH3, CH4). Some isotypes can also include a hingeregion (e.g., IgG).

In certain embodiments, the fusion proteins of the invention are thealpha and beta subunits of FSH each respectively conjugated to aneonatal Fc receptor (FcRn) binding partner. An FcRn binding partner isany molecule that can be specifically bound by the FcRn receptor withconsequent active transport by the FcRn receptor of the FcRn bindingpartner. The FcRn receptor has been isolated from several mammalianspecies including humans. The sequences of the human FcRn, rat FcRn,monkey FcRn, and mouse FcRn are known (Story et al. (1994) J. Exp. Med.180:2377). The FcRn receptor binds IgG (but not other immunoglobulinclasses such as IgA, IgM, IgD, and IgE) at relatively low pH, activelytransports the IgG transcellularly in a luminal to serosal direction,and then releases the IgG at relatively higher pH found in theinterstitial fluids. It is expressed in adult epithelial tissue (U.S.Pat. Nos. 6,030,613 and 6,086,875) including lung and intestinalepithelium (Israel et al. (1997) Immunology 92:69), renal proximaltubular epithelium (Kobayashi et al. (2002) Am. J. Physiol. RenalPhysiol. 282:F358), as well as nasal epithelium, vaginal surfaces, andbiliary tree surfaces.

An FcRn binding partner contains a ligand for the FcRn which mimics thatportion of the Fc domain of IgG which binds the FcRn (i.e., an Fc, an Fcdomain, Fc fragment, Fc fragment homolog, and Fc mimetic). Thus the FcRnbinding partners of the present invention encompass any molecule thatcan be specifically bound by the FcRn receptor including whole IgG, theFc fragment of IgG, and other fragments that include the completebinding region of the FcRn receptor. In certain embodiments, the FcRnbinding partner is non-specific IgG or an FcRn-binding fragment of IgG.Most typically the FcRn binding partner corresponds to the Fc fragmentof IgG, i.e., Fcγ. The Fcγ can be native or it can be modified so thatit has a higher affinity for FcRn than native Fcγ. Such modification caninclude substitution of certain amino acid residues involved in contactwith FcRn. The region of the Fc portion of IgG that binds to the FcRnreceptor has been described based on X-ray crystallography (Burmeisteret al. (1994) Nature 372:379). The major contact area of the Fc with theFcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contactsare all within a single Ig heavy chain. The major contact sites includeamino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436of the CH3 domain. References made to amino acid numbering ofimmunoglobulins or immunoglobulin fragments, or regions, are all basedon Kabat et al. 1991, Sequences of Proteins of Immunological Interest,U.S. Department of Public Health, Bethesda, Md. The Fcγ can be modifiedso that it has a longer circulating half-life than native Fcγ. Suchmodification can include substitution of certain amino acid residuesinvolved in interaction with Fc receptors other than FcRn, substitutionof certain amino acid residues involved in glycosylation, and the like.Specific exemplary modifications are described hereinbelow.

In other embodiments of the invention the FcRn binding partner is an Fcfragment, meaning an immunoglobulin heavy chain constant region, portionthereof, or variant thereof. It can be, or be derived from, animmunoglobulin heavy chain constant region, including, but not limitedto a human immunoglobulin heavy chain constant region, a non-humanprimate immunoglobulin heavy chain constant region, a bovineimmunoglobulin heavy chain constant region, a porcine immunoglobulinheavy chain constant region, a murine immunoglobulin heavy chainconstant region, an ovine immunoglobulin heavy chain constant region, ora rat immunoglobulin heavy chain constant region.

The Fc fragments or FcRn binding partners of the invention can includethe entire heavy chain constant region, or a fragment or analog thereof.A heavy chain constant region can comprise a CH1 domain, a CH2 domain, aCH3 domain, a CH4 domain, and/or a hinge region. In one embodiment aheavy chain constant region can comprise a hinge region, a CH2 domain,and a CH3 domain.

The immunoglobulin can be produced recombinantly or synthetically. Theimmunoglobulin can be isolated from a cDNA library. The immunoglobulincan be isolated from a phage library (see McCafferty et al. (1990)Nature 348:552). The immunoglobulin can be obtained by gene shuffling ofknown sequences (Mark et al. (1992) Bio/Technol. 10:779). Theimmunoglobulin can be isolated by in vivo recombination (Waterhouse etal. (1993) Nucl. Acid Res. 21:2265). The immunoglobulin can be ahumanized immunoglobulin (Jones et al. (1986) Nature 332:323).

An Fc fragment can be comprised of the CH2 and CH3 domains of animmunoglobulin and optionally the hinge region of the immunoglobulin.The Fc fragment can be of an IgG, an IgA, an IgM, an IgD, an IgE. In oneembodiment, the immunoglobulin is an IgG, IgA or IgD. In one embodimentthe Fc fragment is an Fc fragment of IgG. The Fc fragment can be the Fcfragment of an IgG1, an IgG2, an IgG3 or an IgG4. In one embodiment, theimmunoglobulin is human IgG1. In yet another embodiment, theimmunoglobulin is IgG2. In another embodiment, the Fc fragment iscomprised of the amino acid sequence from amino acid number 145 to aminoacid number 371 of SEQ ID NO:4, or an analog thereof. In one embodimentthe Fc fragment is encoded by a nucleic acid sequence comprisingnucleotides 446 to 1126 of SEQ ID NO.3.

The FcRn binding partners or Fc fragments can include an Fc variant. Fcvariant refers to a molecule or sequence that is modified from a nativeFc but still comprises a binding site for the salvage receptor, FcRn (WO97/34631). Native refers to an Fc that has not been modified by a human.WO 96/32478 describes exemplary Fc variants, as well as interaction withthe salvage receptor. Thus, the term “Fc variant” in one embodimentcomprises a molecule or sequence that is humanized from a non-humannative Fc. Furthermore, a native Fc comprises sites that may be removedbecause they provide structural features or biological activity that arenot required for the fusion molecules of the present invention. Thus, Fcvariant comprises a molecule or sequence that lacks one or more nativeFc sites or residues that affect or are involved in (1) disulfide bondformation, (2) incompatibility with a selected host cell (3) N-terminalheterogeneity upon expression in a selected host cell, (4)glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC).

The Fc region of IgG can be modified according to well recognizedprocedures such as site directed mutagenesis and the like to yieldmodified IgG or Fc fragments or portions thereof that will be bound byFcRn. Such modifications include modifications remote from the FcRncontact sites as well as modifications within the contact sites thatpreserve or even enhance binding to the FcRn. For example the followingsingle amino acid residues in human IgG1 Fc (Fcγ1) can be substitutedwithout significant loss of Fc binding affinity for FcRn: P238A, S239A,K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A,E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A,N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A,Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A,E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, A330S, P331A,P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A,Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375AD376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A,Y391 F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A,N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A,and K447A, where for example P238A represents wildtype prolinesubstituted by alanine at position number 238. In addition to alanine,other amino acids may be substituted for the wildtype amino acids at thepositions specified above. Mutations may be introduced singly into Fc,giving rise to more than one hundred FcRn binding partners distinct fromnative Fc. Additionally, combinations of two, three, or more of theseindividual mutations may be introduced together, giving rise to hundredsmore FcRn binding partners.

Certain of the above mutations may confer new functionality upon theFcRn binding partner. For example, one embodiment incorporates N297A,removing a highly conserved N-glycosylation site. The effect of thismutation is to reduce binding to immune effector cells and potentiallydecrease immunogenicity, thereby enhancing circulating half-life of theFcRn binding partner, and to render the FcRn binding partner incapableof binding to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA, withoutcompromising affinity for FcRn (Routledge et al. (1995) Transplantation60:847; Friend et al. (1999) Transplantation 68:1632; Shields et al.(1995) J. Biol. Chem. 276:6591). Additionally, at least three human Fcgamma receptors appear to recognize a binding site on IgG within thelower hinge region, generally amino acids 234-237. Therefore, anotherexample of new functionality and potential decreased immunogenicity mayarise from mutations of this region, as for example by replacing aminoacids 233-236 of human IgG1 “ELLG” with the corresponding sequence fromIgG2 “PVA” (with one amino acid deletion). It has been shown that FcγRI,FcγRII, and FcγRIII, which mediate various effector functions, will notbind to IgG1 when such mutations have been introduced (Ward and Ghetie(1995) Therapeutic Immunology 2:77 and Armour et al. (1999) Eur. J.Immunol. 29:2613). As a further example of new functionality arisingfrom mutations described above, affinity for FcRn may be increasedbeyond that of wildtype in some instances. This increased affinity mayreflect an increased “on” rate, a decreased “off” rate, or both anincreased “on” rate and a decreased “off” rate. Mutations believed toimpart an increased affinity for FcRn include T256A, T307A, E380A, andN434A (Shields et al. (2001) J. Biol. Chem. 276:6591).

In certain embodiments the FcRn binding partner or Fc fragment is apolypeptide including the sequence PKNSSMISNTP (SEQ ID NO:11) andoptionally further including a sequence selected from HQSLGTQ (SEQ IDNO:12), HQNLSDGK (SEQ ID NO:13), HQNISDGK (SEQ ID NO:14), or VISSHLGQ(SEQ ID NO:15; U.S. Pat. No. 5,739,277).

Two FcRn receptors can bind a single Fc molecule. Crystallographic datasuggest that each FcRn molecule binds a single polypeptide of the Fchomodimer. Linking the FcRn binding partner, e.g., an Fc fragment of anIgG, to heterodimeric FSH thus provides a means of delivering FSHorally, nasally, via an ocular route, or via a pulmonary route. Fordelivery nasally or via a pulmonary route, in one embodiment the fusionprotein is administered as an aerosol.

The skilled artisan will understand that portions of an immunoglobulinconstant region for use in the fusion protein of the invention caninclude mutants or analogs thereof, or can include chemically modifiedimmunoglobulin constant regions or fragments thereof (e.g., pegylation)(see, e.g., Aslam and Dent (1998) Bioconjugation: Protein CouplingTechniques for the Biomedical Sciences, Macmillan Reference, London). Inone instance a mutant can provide for enhanced binding of an FcRnbinding partner for the FcRn. Also contemplated for use in the fusionprotein of the invention are peptide mimetics of at least a portion ofan immunoglobulin constant region, e.g., a peptide mimetic of an Fcfragment or a peptide mimetic of an FcRn binding partner. In oneembodiment, the peptide mimetic is identified using phage display (See,e.g., McCafferty et al. (1990) Nature 348:552; Kang et al. (1991) Proc.Natl. Acad. Sci. USA 88:4363; EP 0 589 877 B1).

4. Optional Linkers

The fusion protein of the invention can optionally comprise at least onelinker molecule. In one embodiment, the linker is comprised of aminoacids linked together by peptide bonds, wherein the amino acids areselected from the twenty naturally occurring amino acids. In variousembodiments the linker can comprise 1-5 amino acids, 1-10 amino acids,1-20 amino acids, 10-50 amino acids, 50-100 amino acids, or 100-200amino acids. In one embodiment the amino acids are selected fromglycine, alanine, proline, asparagine, glutamine, and lysine. In oneembodiment a linker is made up of a majority of amino acids that aresterically unhindered, such as glycine and alanine.

The linker in one embodiment can comprise the sequence Gn (equivalently,-(Gly)n-). The linker can in one embodiment comprise the sequence (GGS)nor (GGGGS)n. In each instance, n may be an integer, such as 1, 2, 3, 4,5, 6, 7, 8, 9, or 10. Examples of linkers include, but are not limitedto, GGG, SGGSGGS (SEQ ID NO:16), GGSGGSGGSGGSGGG (SEQ ID NO:17),GGSGGSGGSGGSGGSGGS (SEQ ID NO:18), and GGGGSGGGGSGGGGS (SEQ ID NO:10;GS15).

In one embodiment the linker is an 8-amino acid linker EFAGAAAV (SEQ IDNO:9).

Non-peptide linkers are also possible. For example, alkyl linkers suchas —NH—(CH2)m-C(O)—, wherein m=2-20, could be used. These alkyl linkersmay further be substituted by any non-sterically hindering group such aslower alkyl (e.g., C1 to C6) lower acyl, halogen (e.g., Cl, Br), CN,NH2, phenyl, etc. An exemplary non-peptide linker is a PEG linker.Additional linkers useful according to the present invention aredescribed in U.S. Pat. No. 6,660,843.

C. Nucleic Acid Constructs

The invention also relates to nucleic acid constructs encoding theheterodimer FSH-Fc fusion proteins of the invention. Each nucleic acidsequence comprises a first nucleic acid sequence encoding one subunit ofFSH, for example the alpha subunit of FSH, operatively linked to asecond nucleic acid sequence encoding at least an Fc fragment or FcRnbinding partner. In one embodiment a first nucleic acid sequenceencoding the alpha subunit of FSH is operatively linked to a secondnucleic acid sequence encoding at least an Fc fragment or FcRn bindingpartner, and a third nucleic acid sequence encoding the beta subunit ofFSH is operatively linked to a fourth nucleic acid sequence encoding atleast an Fc fragment or FcRn binding partner (second and fourth nucleicacid sequences generally being identical). The nucleic acids of theinvention thus pertain to nucleic acid constructs encoding bothpolypeptide chains of the subject fusions. The nucleic acids of theinvention can be present in a single nucleic acid construct or inseparate constructs. The nucleic acid sequence can also includeadditional sequences or elements known in the art (e.g., promoters,enhancers, poly A sequences, signal sequence). The nucleic acid sequencecan optionally include a sequence encoding a linker placed between thenucleic acid sequence encoding the alpha or beta subunits of FSH and thenucleic acid sequence encoding the Fc fragment or FcRn binding partner.

In one embodiment, each nucleic acid construct is comprised of DNA. Inanother embodiment, each nucleic acid construct is comprised of RNA. Thenucleic acid construct can be a vector, e.g., a viral vector or aplasmid. Examples of viral vectors include, but are not limited toadenovirus vector, an adeno associated virus vector, and murine leukemiavirus vector. Examples of plasmids include but are not limited to, e.g.,pUC, pGEX, pcDNA3, pcDNA4, pcDNA6, and pED.dC. In certain embodimentsthe plasmid is an expression plasmid.

In one embodiment, the nucleic acid construct comprises the nucleic acidsequences of SEQ ID NO:1 and SEQ ID NO:3.

Due to the known degeneracy of the genetic code, wherein more than onecodon can encode the same amino acid, a DNA sequence can vary from thatshown in SEQ ID NOS:1 and 3 and still encode a polypeptide having theamino acid sequence of SEQ ID NOS:2 and 4, respectively. Such variantDNA sequences can result from silent mutations (e.g., occurring duringPCR amplification), or can be the product of deliberate mutagenesis of anative sequence. The invention thus provides isolated DNA sequencesencoding polypeptides of the invention, selected from: (a) DNAcomprising the nucleotide sequences of SEQ ID NO:1 and 3; (b) DNAencoding the polypeptide of SEQ ID NO:2 and 4; (c) DNA capable ofhybridization to a DNA of (a) or (b) under conditions of moderatestringency and which encodes polypeptides of the invention; (d) DNAcapable of hybridization to a DNA of (a) or (b) under conditions of highstringency and which encodes polypeptides of the invention, and (e) DNAwhich is degenerate as a result of the genetic code to a DNA defined in(a), (b), (c), or (d) and which encode polypeptides of the invention. Ofcourse, polypeptides encoded by such DNA sequences are encompassed bythe invention.

In another embodiment, the nucleic acid molecules of the invention alsocomprise nucleotide sequences that are at least 80% identical to anative sequence. Also contemplated are embodiments in which a nucleicacid molecule comprises a sequence that is at least 90% identical, atleast 95% identical, at least 98% identical, at least 99% identical, orat least 99.9% identical to a native sequence. In this context thenative sequence can refer to the sequence of Fc, αFSH, or βFSH, as theymay be found in nature. The percent identity may be determined by visualinspection and mathematical calculation. Alternatively, the percentidentity of two nucleic acid sequences can be determined by comparingsequence information using the GAP computer program, version 6.0described by Devereux et al. (1984) Nucl. Acids Res. 12:387, andavailable from the University of Wisconsin Genetics Computer Group. Thepreferred default parameters for the GAP program include: (1) a unarycomparison matrix (containing a value of 1 for identities and 0 for nonidentities) for nucleotides, and the weighted comparison matrix ofGribskov and Burgess (1986) Nucl. Acids Res. 14:6745, as described bySchwartz and Dayhoff, eds., 1979, Atlas of Protein Sequence andStructure, National Biomedical Research Foundation, pp. 353-358; (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps. Other programs usedby one skilled in the art of sequence comparison may also be used.

D. Synthesis of Fusion Proteins

Fusion proteins of the subject invention can be synthesized usingtechniques well known in the art. For example fusion proteins of theinvention can be synthesized recombinantly in cells (see, e.g., Sambrooket al. (1989) Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory, N.Y., and Ausubel et al. (1989) Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley Interscience,N.Y.).

DNA sequences encoding immunoglobulins, or fragments thereof, or FSH, orfragments thereof, may be cloned from a variety of genomic, cDNA, or RNAlibraries known in the art. The techniques for isolating such DNAsequences using probe-based methods are conventional techniques and arewell known to those skilled in the art. Probes for isolating such DNAsequences may be based on published DNA sequences (see, for example,Hieter et al. (1980) Cell 22: 197-207). The polymerase chain reaction(PCR) method disclosed by Mullis et al. (U.S. Pat. No. 4,683,195) andMullis (U.S. Pat. No. 4,683,202) may be used. The choice of library andselection of probes for the isolation of such DNA sequences is withinthe level of ordinary skill in the art. Alternatively, DNA sequencesencoding immunoglobulins, or fragments thereof, or FSH can be obtainedfrom vectors known in the art to contain immunoglobulins, or fragmentsthereof, or FSH.

For recombinant production, a polynucleotide sequence encoding thefusion protein is inserted into an appropriate expression vehicle, i.e.,a vector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation.The nucleic acid encoding the fusion protein is inserted into the vectorin proper reading frame.

The expression vehicle is then transfected or otherwise introduced intoa suitable target cell which will express the protein, e.g., a fusionprotein. Transfection techniques known in the art include, but are notlimited to, liposomal transfection, calcium phosphate precipitation(Wigler et al. (1978) Cell 14:725), and electroporation (Neumann et al.(1982) EMBO J. 1:841). A variety of host-expression vector systems maybe utilized to express the fusion proteins described herein ineukaryotic cells. In one embodiment, the eukaryotic cell is an animalcell, including mammalian cells (e.g., CHO, BHK, COS, HeLa cells). Whenthe fusion protein is expressed in a eukaryotic cell, the DNA encodingthe fusion protein may also code for a signal sequence that will permitthe fusion protein to be secreted. One skilled in the art willunderstand that while the protein is translated the signal sequence iscleaved by the cell to form the mature fusion protein. Various signalsequences are known in the art, e.g., mouse Igκ light chain signalsequence. Alternatively, where a signal sequence is not included, thefusion protein can be recovered by lysing the cells.

The fusion protein of the invention can be synthesized in a transgenicanimal, such as a rodent, goat, sheep, pig, or cow. The term “transgenicanimals” refers to non-human animals that have incorporated a foreigngene into their genome. Because this gene is present in germlinetissues, it is passed from parent to offspring. Exogenous genes areintroduced into single-celled embryos (Brinster et al. (1985) Proc.Natl. Acad. Sci. USA 82:4438). Methods of producing transgenic animalsare known in the art, including transgenics that produce immunoglobulinmolecules (Wagner et al. (1981) Proc. Natl. Acad. Sci. USA 78:6376;McKnight et al. (1983) Cell 34:335; Brinster et al. (1983) Nature306:332; Ritchie et al. (1984) Nature 312:517; Baldassarre et al. (2003)Theriogenology 59:831; Robl et al. (2003) Theriogenology 59:107;Malassagne et al. (2003) Xenotransplantation 10(3):267).

The expression vectors can encode for tags that permit for easypurification or identification of the recombinantly produced protein.Examples include, but are not limited to, vector pUR278 (Ruther et al.(1983) EMBO J. 2:1791) in which the fusion protein described hereincoding sequence may be ligated into the vector in frame with the lac zcoding region so that a hybrid protein is produced; pGEX vectors may beused to express proteins with a glutathione S-transferase (GST) tag.These proteins are usually soluble and can easily be purified from cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The vectors include cleavage sites (e.g.,PreCission Protease™ (Pharmacia, Peapack, N.J.) for easy removal of thetag after purification. Additional tags include FLAG, a histidine tag(see Example 1 below) a maltose binding protein tag, and others known tothose of skill in the art.

To increase efficiency of production, the polynucleotide can be designedto encode multiple units of the fusion protein of the inventionseparated by enzymatic cleavage sites. The resulting polypeptide can becleaved (e.g., by treatment with the appropriate enzyme) in order torecover the polypeptide units. This can increase the yield ofpolypeptides driven by a single promoter. When used in appropriate viralexpression systems, the translation of each polypeptide encoded by themRNA is directed internally in the transcript, e.g., by an internalribosome entry site (IRES). Thus, the polycistronic construct directsthe transcription of a single, large polycistronic mRNA which, in turn,directs the translation of multiple, individual polypeptides. Thisapproach eliminates the production and enzymatic processing ofpolyproteins and may significantly increase yield of a polypeptidedriven by a single promoter.

Vectors used in transformation and transfection will usually contain aselectable marker used to identify transformants and transfectants. Inbacterial systems this can include an antibiotic resistance gene such asampicillin, blasticidin or kanamycin. Selectable markers for use incultured mammalian cells include genes that confer resistance to drugs,such as neomycin, hygromycin, blasticidin, geneticin, zeocin, andmethotrexate. The selectable marker may be an amplifiable selectablemarker. One amplifiable selectable marker is the dihydrofolate reductasegene (DHFR gene) or the cDNA thereof (Simonsen and Levinson (1983) Proc.Natl. Acad. Sci. USA 80:2495). Selectable markers are reviewed by Thilly(1986) Mammalian Cell Technology, Butterworth Publishers, Stoneham,Mass., and the choice of selectable markers is well within the level ofordinary skill in the art.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, U.S. Pat. No.4,713,339).

The expression elements of the expression systems vary in their strengthand specificities. Depending on the host/vector system utilized, any ofa number of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedron promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g. heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g. the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genome of mammalian cells (e.g.metallothionein promoter) or from mammalian viruses (e.g. the adenoviruslate promoter; the vaccinia virus 7.5 K promoter, the CMV promoter) maybe used; when generating cell lines that contain multiple copies ofexpression product, SV40-, BPV- and EBV-based vectors may be used withan appropriate selectable marker.

In cases where plant expression vectors are used, the expression ofsequences encoding linear or non-cyclized forms of the fusion proteinsof the invention may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S RNA and 19S RNA promoters ofCaMV (Brisson et al. (1984) Nature 310:511-514), or the coat proteinpromoter of TMV (Takamatsu et al. (1987) EMBO J. 6:307-311) may be used;alternatively, plant promoters such as the small subunit of RUBISCO(Coruzzi et al. (1984) EMBO J. 3:1671-1680; Broglie et al. (1984)Science 224:838-843) or heat shock promoters, e.g., soybean hsp17.5-E orhsp17.3-B (Gurley et al. (1986) Mol. Cell. Biol. 6:559-565) may be used.These constructs can be introduced into plant cells using Ti plasmids,Ri plasmids, plant virus vectors, direct DNA transformation,microinjection, electroporation, etc. For reviews of such techniquessee, e.g., Weissbach & Weissbach (1988) Methods for Plant MolecularBiology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson &Corey (1988) Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

In one insect expression system that may be used to produce the fusionproteins of the invention, Autographa californica nuclear polyhidrosisvirus (AcNPV) is used as a vector to express the foreign genes. Thevirus grows in Spodoptera frugiperda cells. A coding sequence may becloned into non-essential regions (for example the polyhedron gene) ofthe virus and placed under control of an AcNPV promoter (for example,the polyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e. virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed (see, e.g., Smith et al. (1983) J. Virol. 46:584; U.S.Pat. No. 4,215,051). Further examples of this expression system may befound in Ausubel et al., eds (1989) Current Protocols in MolecularBiology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

Another system which can be used to express the fusion proteins of theinvention is the glutamine synthetase gene expression system, alsoreferred to as the “GS expression system” (Lonza Biologics PLC,Berkshire UK). This expression system is described in detail in U.S.Pat. No. 5,981,216.

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This fusion gene may then be inserted in theadenovirus genome by in vitro or in vivo recombination. Insertion in anon-essential region of the viral genome (e.g. region E1 or E3) willresult in a recombinant virus that is viable and capable of expressing apolypeptide peptide in infected hosts (see, e.g., Logan & Shenk (1984)Proc. Natl. Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia7.5 K promoter may be used (see, e.g., Mackett et al. (1982) Proc. Natl.Acad. Sci. USA 79:7415; Mackett et al. (1984) J. Virol. 49:857; Panicaliet al. (1982) Proc. Natl. Acad. Sci. USA 79:4927).

Host cells containing DNA constructs of the fusion protein are grown inan appropriate growth medium. As used herein, the term “appropriategrowth medium” means a medium containing nutrients required for thegrowth of cells. Nutrients required for cell growth may include a carbonsource, a nitrogen source, essential amino acids, vitamins, minerals andgrowth factors. For example, the media can contain bovine calf serum orfetal calf serum. The growth medium will generally select for cellscontaining the DNA construct by, for example, drug selection ordeficiency in an essential nutrient, which is complemented by theselectable marker on the DNA construct or co-transfected with the DNAconstruct. Cultured mammalian cells are generally grown in commerciallyavailable serum-containing or serum-free media (e.g. MEM, DMEM).Selection of a medium appropriate for the particular cell line used iswithin the level of ordinary skill in the art.

The recombinantly produced fusion protein of the invention can beisolated from the culture media using procedures well-established in theart (e.g., affinity chromatography, size exclusion chromatography, ionexchange chromatography). The fusion protein of the invention can beisolated from the culture media by column chromatography, e.g., aprotein A column, or by ion exchange chromatography. The culture mediumfrom appropriately grown transformed or transfected host cells isseparated from the cell material, and the presence of fusion proteins isdemonstrated. One method of detecting the fusion proteins, for example,is by the binding of the fusion proteins or portions of the fusionproteins to a specific antibody recognizing the fusion protein of theinvention (e.g., an anti-Fc antibody). An anti-fusion protein antibodymay be a monoclonal or polyclonal antibody raised against the fusionprotein in question. For example, the fusion protein can contain aportion of an immunoglobulin constant region. Antibodies recognizing theconstant region of many immunoglobulins are known in the art and arecommercially available. An antibody can be used to perform an ELISA or awestern blot to detect the presence of the fusion protein of theinvention.

E. Methods of Using Fusion Proteins

The fusion proteins of the invention have many uses as will berecognized by one skilled in the art, including, but not limited tomethods of treating a subject having a reproductive disorder and methodsof treating a subject in need of increased fertility and/or FSH therapyfor treatment of an FSH abnormality.

1. Methods of Treating a Subject Having a Reproductive Disorder

The invention relates to a method of treating a subject having areproductive disorder or FSH abnormality. Such abnormalities aredescribed, for example, in Harrison's Principles of Internal Medicine,15^(th) Ed., E. Braunwald et al., eds, McGraw-Hill, New York, 2001. Thusthe invention provides a method for treating a disease state responsiveto FSH therapy by administering a fusion protein provided herein. Suchtherapies generally relate to infertility or a reproductive disorder.Accordingly, the invention provides a method of increasing a subject'sfertility by administering an amount of the present fusion proteinsufficient to enhance the subject's fertility. In one embodiment, thismethod is used to enhance the efficacy of in vitro fertilizationprotocols. For example, the subject fusion proteins of the instantinvention can enhance the success of in vitro fertilization bystimulating follicular maturation and egg production in a patient. Theinvention also provides a method of increasing a subject's eggproduction or a method of increasing spermatogenesis, by administeringto a subject an effective amount of the instant fusion protein to obtainthe desired increase.

As used herein, a subject can be a mammal, for example, a human, anon-human primate, a horse, a sheep, a cow, a pig, a dog, a cat, or arodent. In one embodiment, the subject is a human.

In one embodiment of the invention is provided a method of increasingthe half-life of heterodimeric FSH. By way of this embodiment, FSH maybe administered to a subject less frequently than current methods allow.For example, an FSH fusion of the invention may be administered onlyone, two, or three times over an 8 to 12 day cycle.

There are numerous assays available by which to determine the activityof a given heterodimeric FSH-Fc construct of the invention and itsusefulness in the above-described methods of treatment. Standard assaysare listed hereinbelow and some are described in detail, withmodifications to certain of the protocols, in the ensuing Examples.

Standard in vitro assays include: rat Sertoli cell bioassays to measureestrogen production (Dorrington, J H and Armstrong, D T (1975) Proc.Natl. Acad. Sci. USA 72:2677); rat Sertoli cell bioassays to measurearomatase activity (Padmanabhan V, Chappel S C, Beitins, I Z (1987)Endocrinology 121:1089-1098); rat granulosa cell bioassay to measurearomatase activity (Jia X C and Hsueh A J W (1986) Endocrinology119:1570-1577; Dahl K D, et al. (1987) J Clin Endocrinol Metab64:486-493); and FSH receptor binding/cAMP production assays (Tano M,Minegishi T, Nakamura K, Karino S, Ibuki Y (1995) Fertil Steril64:1120-1124).

Standard in vivo assays include: in rodents, ovarian weight gain, knownas the Steelman Pohley assay (Steelman S L and Pohley F M (1953)Endocrinology 53:604-616) and testis weight gain (Meachem S J, McLachlanR I, deKretser D M, Robertson D M, Wreford N G (1996) Biol Reprod54:36-44); in non-human primates, estrogen production (Klein J et al.(2002) Fertil Steril 77:1248-1255) and inhibin levels (Weinbauer G F etal. (1994) J Endocrinol 141:113-121); and in humans, serum estrogen,serum inhibin and follicle size/number (Porchet H C et al. (1994) FertilSteril 61:687-695).

The fusion protein of the invention can be administered intravenously,subcutaneously, intra-muscularly, or via any mucosal surface, e.g.,orally, sublingually, buccally, nasally, rectally, vaginally or viapulmonary route. The fusion protein can be implanted within or linked toa biopolymer solid support that allows for the slow release of thefusion protein. In one embodiment the fusion protein is administeredparenterally. In one embodiment the fusion protein is administeredorally. In one embodiment the fusion protein is administered via apulmonary route.

The dose of the fusion protein of the invention will vary depending onthe subject and upon the particular route of administration used.Dosages can range from 0.1 to 100,000 μg/kg body weight. In oneembodiment, the dosing range is 0.1-1,000 μg/kg. The protein can beadministered continuously or at specific timed intervals. In vitroassays may be employed to determine optimal dose ranges and/or schedulesfor administration. In vitro assays that measure FSH activity are knownin the art as described in detail above. Additionally, effective dosesmay be extrapolated from dose-response curves obtained from animalmodels, e.g., non-human primates.

The invention also relates to a pharmaceutical composition comprisingthe subject fusion protein and a pharmaceutically acceptable carrier orexcipients. Examples of suitable pharmaceutical carriers are describedin Remington's Pharmaceutical Sciences by E. W. Martin. Examples ofexcipients can include starch, glucose, lactose, sucrose, gelatin, humanserum albumin, detergent (e.g. Tween-20), malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, polyethylene glycol, propyleneglycol, water, ethanol, and the like. The composition can also containpH buffering reagents, and wetting or emulsifying agents.

For oral administration, the pharmaceutical composition can take theform of tablets or capsules prepared by conventional means. Thecomposition can also be prepared as a liquid, for example a syrup or asuspension. The liquid can include suspending agents (e.g. sorbitolsyrup, cellulose derivatives or hydrogenated edible fats), emulsifyingagents (lecithin or acacia), non-aqueous vehicles (e.g. almond oil, oilyesters, ethyl alcohol, or fractionated vegetable oils), andpreservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid).The preparations can also include flavoring, coloring and sweeteningagents. Alternatively, the composition can be presented as a dry productfor constitution with water or another suitable vehicle.

For buccal administration, the composition may take the form of tabletsor lozenges according to conventional protocols.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of a nebulizedaerosol with or without excipients or in the form of an aerosol sprayfrom a pressurized pack or nebulizer, with optionally a propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator can be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch. Aerosols containing fusion proteins of the subjectinvention may be prepared as described in U.S. 2003/023553 A1 which isincorporated herein by reference.

The pharmaceutical composition can be formulated for parenteral (e.g.intravenous or intramuscular) administration by injection or infusion.Formulations for injection or infusion can be presented in unit dosageform, e.g., in ampoules or in multidose containers with an addedpreservative. The compositions can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., pyrogen-free water.

The pharmaceutical composition can also be formulated for rectaladministration as a suppository or retention enema, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

EXAMPLES Example 1 Construction of Single-Chain FSH-Fc and HeterodimerFSH-Fc Fusion Molecules

For single-chain FSH-Fc constructs, FSH beta was isolated with itsnative signal sequence from a human pituitary mRNA library (Clontech,Palo Alto, Calif.) using standard reverse transcriptase-polymerase chainreaction (RT-PCR) techniques. FSH alpha was isolated from the same humanpituitary mRNA library but without its signal sequence. The two FSHsubunits were ligated to form a contiguous FSH beta-FSH alpha fusionwithout a 3′ termination codon. The Fc fragment of human IgG₁ (hinge,CH2 and CH3 domains; amino acids 221-447, EU numbering) was prepared aspreviously described (U.S. 2003/0235536 A1) using standard polymerasechain reaction (PCR) techniques. Primers were designed to create an8-amino acid linker sequence (EFAGAAAV; SEQ ID NO:9) on the 5′ end ofthe Fc fragment. The human Fc PCR fragment was cloned into the mammalianexpression vector, pED.dC (Genetics Institute, Cambridge, Mass.) thatcontains an adenovirus major late promoter and a mouse dihydrofolatereductase (dhfr) gene as a selectable marker. The single-chain FSHmolecule was then cloned into pED.dC containing the human Fc sequence,thus creating a fusion molecule of FSH beta-FSH alpha-Fc with an eightamino acid linker connecting the FSH subunits to the Fc (FIG. 1 a).

For heterodimer FSH-Fc constructs, both FSH alpha and FSH beta subunitswere isolated from the same human pituitary mRNA library, each with itsnative signal sequence. The 8-amino acid linker sequence from the Fc inpED.dC was replaced by a fifteen amino acid linker sequence, GS15((GGGGS)₃; SEQ ID NO:10). The adenovirus major late promoter in pED.dCwas replaced by a cytomegalovirus (CMV) promoter using standard PCRtechniques to create restriction sites on the 5′ and 3′ ends of the CMVpromoter that allowed excision of the adenovirus major late promoter andreplacement with the CMV promoter. The template for the PCR reaction waspcDNA6N5-His (Invitrogen, Carlsbad, Calif.), a mammalian expressionvector containing a CMV promoter and blasticidin gene for selectionpurposes. The GS15 linker/Fc fragment from pED.dC was also ligated intopcDNA6N5-His to create a second vector with a unique selection marker.FSH alpha was then cloned into the GS15-Fc-pED.dC vector, while FSH betawas cloned into the GS15-Fc-pcDNA6N5-His. For purification purposes a6His tag sequence was fused onto the 3′ end of the Fc in FSH beta-Fc inpcDNA6N5-His (FIG. 1 b).

Resulting full length nucleotide coding and amino acid sequences for FSHalpha-Fc are provided as SEQ ID NO:1 and SEQ ID NO:2, respectively.cctgcaggccaccatggattactacagaaaatatgcagctatctttctggtcacattgtcggtgtttctgcatgttctccSEQ ID NO: 1attccgctcctgatgtgcaggattgcccagaatgcacgctacaggaaaacccattcttctcccagccgggtgccccaatacttcagtgcatgggctgctgcttctctagagcatatcccactccactaaggtccaagaagacgatgttggtccaaaagaacgtcacctcagagtccacttgctgtgtagctaaatcatataacagggtcacagtaatggggggtttcaaagtggagaaccacacggcgtgccactgcagtacttgttattatcacaaatctggtggaggcggatccggtggaggcgggtccggcggtggagggagcgacaaaactcacacgtgcccgccgtgcccagctccggaactgctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

In SEQ ID NO:1 shown above, the signal sequence is underlined and codingsequence for the GS15 linker sequence is shown in bold. The 5′ primersequence for FSH alpha was: ggctagcctgcaggccaccatggattactacagaaaatatgc(SEQ ID NO:5). The 3′ primer sequence for FSH alpha was:tccaccggatccgcctccaccagatttgtgataataacaagtact (SEQ ID NO:6).MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQCMG SEQ ID NO: 2CCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In SEQ ID NO:2 shown above, the signal sequence is underlined and thelinker sequence is shown in bold. The Fc fragment is amino acid numbers132-358.

Resulting full length nucleotide coding and amino acid sequences for FSHbeta-Fc with 6His tag are provided as SEQ ID NO:3 and SEQ ID NO:4,respectively.cctgcaggccaccatgaagacactccagtttttcttccttttctgttgctggaaagcaatctgctgcaatagctgtgagSEQ ID NO: 3ctgaccaacatcaccattgcaatagagaaagaagaatgtcgtttctgcataagcatcaacaccacttggtgtgctggctactgctacaccagggatctggtgtataaggacccagccaggcccaaaatccagaaaacatgtaccttcaaggaactggtatacgaaacagtgagagtgcccggctgtgctcaccatgcagattccttgtatacatacccagtggccacccagtgtcactgtggcaagtgtgacagcgacagcactgattgtactgtgcgaggcctggggcccagctactgctcctttggtgaaatgaaagaaggtggaggcggatccggtggaggcgggtccggcggtggagggagcgacaaaactcacacgtgcccgccgtgcccagctccggaactgctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaacatcatcaccatcaccactga

In SEQ ID NO:3 shown above, the signal sequence is underlined, the 6Histag is shown in italics, and the GS15 linker sequence is shown in bold.The 5′ primer sequence for FSH beta was:ggctagcctgcaggccaccatgaagacactccagtttttct (SEQ ID NO:7). The 3′ primersequence for FSH beta was: tccaccggatccgcctccaccttctttcatttcaccaaagga(SEQ ID NO:8). MKTLQFFFLFCCWKAICCNSCELTNITIAIEKEECRFCISINTTWCAGYCYTRDLVSEQ ID NO: 4 YKDPARPKIQKTCTFKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDSTDCTVRGLGPSYCSFGEMKEGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKH HHHHH

In SEQ ID NO:4 shown above, the signal sequence is underlined, the Histag is shown in italics, and the GS15 linker sequence is shown in bold.The Fc fragment is amino acid numbers 145-371.

Example 2 Expression and Purification of Single-Chain FSH-Fc andHeterodimer FSH-Fc

Single-chain FSH-Fc was transfected into CHO DG44 cells lacking adihydrofolate reductase gene using standard Superfect transfectionprotocols (Qiagen, Valencia, Calif.). After 48 h, transfected cells wereselected in MEMα without ribonucleosides and deoxyribonucleosidescontaining 5% dialyzed FBS. To obtain higher protein expression levels,cells were treated with methotrexate at levels up to 200 nM. Forexpression studies, cells were seeded into roller bottles inDMEM:F12+10% FBS and incubated for three days before changing the mediumto DMEM:F12+5 μg/ml human insulin. Conditioned medium was collecteddaily for 10 days and then filtered through 0.2 μm filters and stored at4° C. until purification. Single-chain FSH-Fc was purified from cellsupernatant using standard protein A affinity chromatography. Oncemedium containing single-chain FSH-Fc was loaded, protein A columns werewashed with 5-10 column volumes of PBS (10 mM phosphate pH 7.4, 2.7 mMKCl and 137 mM NaCl) and bound protein eluted with 0.1 M glycine pH 3.0.Eluted single-chain FSH-Fc was then dialyzed into PBS and stored at −80°C. in aliquots containing 10% glycerol. Single-chain FSH-Fc isapproximately 90% pure following a single protein A chromatography step(FIG. 2).

Heterodimer FSH-Fc was expressed by co-transfecting FSH alpha-Fc and FSHbeta-Fc-6His expression vectors into CHO DG44 cells using standardSuperfect transfection methods. Forty-eight hours after transfection,cells were selected in MEMα without ribonucleosides anddeoxyribonucleosides containing 5% dialyzed FBS and 10 μg/ml blasticidinto select only for cells containing both FSH alpha-Fc and FSH beta-Fcexpression vectors. To obtain higher expression levels, cells weretreated with methotrexate up to 50 nM. For expression studies, cellswere seeded into roller bottles and cell supernatant containing secretedprotein collected in the same way as for single-chain FSH-Fc. Since FSHalpha-Fc and FSH beta-Fc were co-transfected, cell supernatantscontained mixtures of FSH alpha-Fc homodimer, FSH beta-Fc homodimer, andheterodimer FSH-Fc that required separation. An initial purificationusing protein A affinity chromatography was performed in the same way asdescribed above for single-chain FSH-Fc. After protein A elution,protein was further purified by nickel affinity chromatography. FSHbeta-Fc homodimers and heterodimer FSH-Fc bind to nickel affinitycolumns due to the presence of a 6His tag. Heterodimer FSH-Fc wasseparated from the FSH beta-Fc homodimer by elution with an imidazolegradient (0-500 mM) with the heterodimer FSH-Fc eluting at approximately30 to 90 mM imidazole. Heterodimer FSH-Fc was approximately 90% purefollowing protein A and nickel affinity chromatography steps (FIG. 2).

FIG. 1 c shows a schematic diagram of single-chain FSH-Fc andheterodimer FSH-Fc fusion proteins. In this example single-chain FSH-Fcis a fusion molecule of FSH alpha, FSH beta, and the Fc portion of ahuman IgG₁ molecule including the hinge, CH2, and CH3 domains. Thus anFc dimer of single-chain FSH-Fc contained two FSH alpha and two FSH betasubunits. In contrast, in this example heterodimer FSH-Fc was made insuch a way that the Fc dimer contained a single FSH alpha subunit on oneFc chain and a single FSH beta subunit on the other Fc chain. Because ofthe extra FSH subunits in the single-chain FSH-Fc molecule, single-chainFSH-Fc protein is larger than the heterodimer FSH-Fc protein when rununder either reducing conditions (approximately 75 kDa compared to 50kDa, respectively) or non-reducing conditions (approximately 150 kDacompared to 100 kDa, respectively) on SDS-PAGE gels (FIG. 2). Undernon-reducing conditions, both single-chain FSH-Fc and heterodimer FSH-Fcpredominantly form Fc dimers (FIG. 1 c and FIG. 2).

Example 3 Rodent Pharmacodynamic Studies: Subcutaneous Dosing

In order to determine whether single-chain FSH-Fc and heterodimer FSH-Fcfusion proteins have similar bioactivity to human recombinant FSH,21-day-old female rats (10 rats per group) were dosed subcutaneouslywith a single dose of recombinant FSH (Follistim, Organon, West Orange,N.J.), single-chain FSH-Fc, or heterodimer FSH-Fc in PBS at 1 nmol/kg.Seventy-two hours after dosing, ovarian weight was measured in each rat.Steelman S L and Pohley F M (1953) Endocrinol 53:604-16. Statistics wereanalyzed using SigmaStat version 2.0 (RockWare, Inc., Golden, Colo.).Results are presented in FIG. 3.

As shown in FIG. 3, ovarian weight was significantly increased in femalerats treated with a single dose of recombinant FSH compared to vehicle(14.3±1.7 mg compared to 12.1±1.0 mg, respectively, p=0.003). However,single-chain FSH-Fc and heterodimer FSH-Fc produced an even greaterincrease in ovarian weight compared to vehicle and FSH treated groups(20.8±3.9 mg and 26.9±6.1 mg, respectively; p<0.001). Heterodimer FSH-Fcwas significantly more active than single-chain FSH-Fc in thisexperiment (p=0.016).

Example 4 Rodent Pharmacokinetic Studies

Neonatal rats express high levels of FcRn in the small intestines duringthe first three weeks of life. This system can therefore be used todetermine oral delivery of FcRn binding molecules such as Fc fusionproteins. Ten-day-old neonatal rats (four rats per group) were dosedorally with 0.3 mg/kg single-chain FSH-Fc or heterodimer FSH-Fc innormal saline containing 5 mg/ml soybean trypsin inhibitor. At varioustimes after dosing, blood was collected by cardiac puncture and serumprepared. Serum was stored at −20° C. until analysis by ELISA. Asandwich ELISA was developed using an anti-FSH coating antibody(Fitzgerald Industries, Concord, Mass.) and a horseradish peroxidaseconjugated anti-Fc detection antibody (Pierce Chemical Company,Rockford, Ill.). The standard curve for the ELISA was created with thesame lot of protein used to dose the rats. Samples were analyzed intriplicate. Pharmacokinetic parameters were estimated using WinNonlinversion 4.1 (Pharsight, Mountain View, Calif.). Results are presented inFIG. 4.

As shown in FIG. 4, high levels of single-chain FSH-Fc and heterodimerFSH-Fc were measured in neonatal rat serum after oral dosing (2.4 μg/mland 3.8 μg/ml, respectively, which are the average maximum serumconcentrations), and both proteins had long terminal half-lives of 60 hand 69 h, respectively. In combination with the bioactivity results inFIG. 3, these data suggest that the higher in vivo activity ofsingle-chain and heterodimer FSH-Fc compared to recombinant FSH may bedue to the long half-lives of the fusion proteins.

Example 5 Role of FcRn in Oral Delivery of Single-Chain FSH-Fc andHeterodimer FSH-Fc in Neonatal Rats

In order to show that oral delivery of single-chain FSH-Fc andheterodimer FSH-Fc is due to FcRn binding and transcytosis, 10-day-oldneonatal rats were orally dosed with a mixture of ¹²⁵I-labeledsingle-chain FSH-Fc or heterodimer FSH-Fc and a 300-fold excess ofunlabeled human IgG₁ (ICN, Irvine, Calif.) in normal saline with 5 mg/mlsoybean trypsin inhibitor. Single-chain FSH-Fc and heterodimer FSH-Fcwere iodinated with ¹²⁵I sodium iodide (Perkin Elmer, Boston, Mass.)using iodobeads (Pierce) according to manufacturers' protocols. Freeiodine was separated from iodinated protein on a PD-10 desalting column.Two hours after dosing, blood was collected by cardiac puncture andserum prepared. A 100 μl aliquot of serum was incubated with protein Atris acrylamide beads (Pierce) at 4° C. for 1 h. Protein A beads werethen washed twice with PBS and eluted with SDS sample buffer containing10% β-mercaptoethanol. Samples were boiled and analyzed on 4-20%SDS-PAGE gels, dried, and quantitation was performed on a StormPhosphorimager (Molecular Dynamics, Piscataway, N.J.). Results arepresented in FIG. 5.

Oral delivery of both single-chain FSH-Fc and heterodimer FSH-Fc wasgreatly reduced in the presence of excess IgG₁ (83% and 53% reduction intransport respectively as determined by phosphorimage analysis). SinceIgG₁ is a natural ligand for FcRn, this suggests that single-chainFSH-Fc and heterodimer FSH-Fc bind specifically to, and are transportedby, FcRn, and the process can be disrupted by the presence of excessIgG₁.

Example 6 Rodent Pharmacodynamic Studies: Oral Dosing

Two-day-old male rats (10 rats per group) were orally dosed with 1nmol/kg recombinant human FSH (Follistim, Organon), single-chain FSH-Fc,or heterodimer FSH-Fc in PBS with 5 mg/ml soybean trypsin inhibitor.Rats were dosed daily for 14 days before the right testis from eachanimal was removed and weighed. Meachem S J et al. (1996) Biol Reprod54:36-44. Statistics were analyzed using SigmaStat version 2.0(RockWare, Inc.). Results are presented in FIG. 6.

As shown in FIG. 6, in this assay recombinant FSH did not produce anincrease in testis weight compared to the vehicle treated group(49.1±8.1 mg compared to 55.4±8.1 mg, respectively), suggesting thatrecombinant FSH was not orally active in this model (FIG. 6 a). Incontrast, single-chain FSH-Fc (FIG. 6 a and FIG. 6 b) and heterodimerFSH-Fc (FIG. 6 b) treatments resulted in significant increases in testisweight compared to vehicle treated animals (113.0±19.8 mg and 139.6±11.9mg compared to 58.6±10.4 mg, respectively; p<0.001). Similarly to thesubcutaneous dosing experiment, heterodimer FSH-Fc was significantlymore active than single-chain FSH-Fc in this experiment (p=0.003).

Example 7 Pharmacokinetic Studies in Cynomolgus Monkeys

We have previously shown expression of FcRn in cynomolgus monkey andhuman lung, and that an erythropoietin-Fc fusion protein is absorbed andretains activity after pulmonary administration (Spiekermann G M et al.(2002) J Exp Med 196: 300-310; Bitonti A J et al. (2004) Proc Natl AcadSci USA 101: 9763-9768). We therefore next wanted to determine whethersingle-chain FSH-Fc and heterodimer FSH-Fc could be dosed through thelung in a non-human primate and retain biological activity.

All studies with cynomolgus monkeys were conducted using approvedprotocols, following NIH guidelines for the care and use of researchanimals. Prior to pulmonary administration, animals were anesthetizedwith a combination of ketamine and Valium and intubated withendotracheal tubes. Aerosols of single-chain FSH-Fc (in PBS pH7.4) andheterodimer FSH-Fc (in PBS pH7.4 with 0.1% human serum albumin) werecreated with an Aeroneb Pro™ nebulizer (Aerogen, Mountain View, Calif.)and administered to cynomolgus monkeys (deposited dose approximately 45μg/kg) through the endotracheal tubes. A Bird Mark 7A respiratorregulated the depth (20-40% vital capacity) and rate (28-30 breaths perminute) of respiration of each monkey such that the delivery ofsingle-chain FSH-Fc and heterodimer FSH-Fc was targeted to the centralairways. Aerosol particle size was approximately 4-5 μm. Blood sampleswere collected at various times after pulmonary dosing and serumprepared. Serum single-chain FSH-Fc and heterodimer FSH-Fc levels werequantified using a commercially available FSH ELISA kit (DRGInternational, Mountainside, N.J.) according to manufacturer'sdirections. The standard curve for each assay was created with the samelot of single-chain FSH-Fc or heterodimer FSH-Fc used to dose themonkeys. Pharmacokinetic parameters were estimated using WinNonlinversion 4.1 (Pharsight). Results are presented in Table 2 and FIG. 7.

TABLE 2. Pharmacokinetic parameter estimates after pulmonary dosing ofsingle-chain FSH-Fc and heterodimer FSH-Fc in cynomolgus monkeys at adeposited dose of approximately 45 μg/kg. single-chain FSH-Fcheterodimer FSH-Fc Parameter Monkey 1 Monkey 2 Monkey 3 Monkey 4 Cmax,ng/ml 91.4 93.9 68.7 130.5 t_(1/2), h 210 54.7 182 219 AUC, 17489 1308626526 53001 hr * ng/ml

Deposited doses of approximately 45 μg/kg of each protein resulted inmaximum serum concentrations of 91 and 94 ng/ml single-chain FSH-Fc and69 and 131 ng/ml heterodimer FSH-Fc (Table 2). The terminal half-life ofboth proteins was measured as 55 and 210 h for single-chain FSH-Fc and182 and 219 h for heterodimer FSH-Fc (Table 2). These half-lives aresignificantly longer than that of recombinant FSH, which isapproximately 24 h in humans (le Cotonnec J-Y et al. (1994) FertilSteril 61:679-686 and in non-human primates (Porchet H C et al. (1993)Drug Metab Dispos 21:144-150; Weinbauer G F et al. (1994) J Endocrinol141:112-121). Thus an advantage of using FSH-Fc fusion proteins intreatment of infertility would be the potential for greatly reduceddosing frequency.

Example 8 Pharmacodynamic Measurements in Cynomolgus Monkeys

Inhibin is a pharmacodynamic marker of FSH activity. Therefore serumsamples obtained after pulmonary dosing of single-chain FSH-Fc andheterodimer FSH-Fc were also used to determine inhibin levels using acommercially available ELISA kit (Diagnostic Systems Laboratories,Webster, Tex.) following manufacturer's instructions. Weinbauer G F etal. (1994) J Endocrinol 141:113-121. Results are presented in FIG. 8.Deposited pulmonary doses of approximately 45 μg/kg single-chain FSH-Fcresulted in maximum inhibin B concentrations of 1 and 1.6 ng/ml,equivalent to 1.2- and 1.4-fold stimulation above baseline levels. Thesame deposited pulmonary dose of heterodimer FSH-Fc resulted in maximuminhibin B concentrations of 2.7 and 7.4 ng/ml, equivalent to 7.1- and5.9-fold stimulation above baseline. Fourteen days after treatment withpulmonary heterodimer FSH-Fc, inhibin B levels had not returned tobaseline.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupercede and/or take precedence over any such contradictory material.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with a true scope and spirit of the invention beingindicated by the following claims.

1. A heterodimeric fusion protein comprising two associated polypeptidechains, the first chain comprising an alpha subunit of folliclestimulating hormone (αFSH) conjugated to an FcRn binding partner and thesecond chain comprising a beta subunit of FSH (βFSH) conjugated to anFcRn binding partner, wherein the head of αFSH is aligned with the headof βFSH and the tails of each of the respective FcRn binding partnersare aligned.
 2. The fusion protein of claim 1, further comprising alinker between the FSH subunits and the FcRn binding partners.
 3. Afusion protein comprising two polypeptide chains of the formulaαFSH-L-Fc:βFSH-L-Fc wherein αFSH is an alpha subunit of FSH, βFSH is abeta subunit of FSH, L is a linker or direct bond, and Fc is an Fcfragment of an immunoglobulin, wherein carboxy termini of αFSH and βFSHare linked either directly or indirectly through L to the amino terminusof the respective Fc, further wherein the colon (:) represents anassociation between the two polypeptide chains of the fusion protein,and further wherein the head of αFSH is aligned with the head of βFSHand the tails of each of the respective Fc fragments are aligned.
 4. Afusion protein comprising two polypeptide chains of the formulaFc-L-αFSH:Fc-L-βFSH wherein αFSH is the alpha subunit of FSH, βFSH isthe beta subunit of FSH, L is a linker or direct bond, and Fc is an Fcfragment of an immunoglobulin, wherein the amino termini of αFSH andβFSH are linked either directly or indirectly through L to the carboxyterminus of the respective Fc, further wherein the colon (:) representsan association between the two polypeptide chains of the fusion protein,and further wherein the head of αFSH is aligned with the head of βFSHand the tails of each of the respective Fc fragments are aligned.
 5. Thefusion protein of any one of claims 1, 3, or 4, wherein FSH is humanFSH.
 6. The fusion protein of claim 3 or 4, wherein the Fc fragmentcomprises an amino acid sequence having at least 80% identity with thesequence set forth by amino acid number 145 to amino acid number 371 ofSEQ ID NO:4.
 7. The fusion protein of claim 3 or 4, wherein the Fcfragment comprises an amino acid sequence having the sequence set forthby amino acid number 145 to amino acid number 371 of SEQ ID NO:4.
 8. Thefusion protein of claim 3 or 4, wherein the Fc is the Fc fragment ofIgG.
 9. The fusion protein of claim 3 or 4, wherein L is a direct bond.10. The fusion protein of any one of claims 2 to 4, wherein said linkeris about 1 to 20 amino acids.
 11. The fusion protein of claim 10,wherein said linker is about 8 to 15 amino acids.
 12. The fusion proteinof claim 11, wherein said linker has the amino acid sequence EFAGAAAV(SEQ ID NO:9).
 13. The fusion protein of claim 10, wherein said linkercomprises a sequence -(Gly)_(n)-, wherein n is an integer from about 1to
 20. 14. The fusion protein of claim 10, wherein said linker comprisesa sequence chosen from -(GGS)_(n)- or -(GGGGS)_(n)-, wherein n is aninteger of about 1 to
 7. 15. The fusion protein of claim 3 or 4, whereinthe association between the two polypeptide chains comprises anassociation between the alpha and beta subunits of FSH.
 16. The fusionprotein of claim 3 or 4, wherein the association between the twopolypeptide chains comprises an association between the two Fcfragments.
 17. The fusion protein of claim 16, wherein the associationcomprises at least one disulfide bond.
 18. A pharmaceutical compositioncomprising the fusion protein of any one of claims 1, 3, or 4, and apharmaceutically acceptable excipient.
 19. A nucleic acid encoding oneor both of the polypeptide chains of the fusion protein of any one ofclaims 1, 3, or
 4. 20. A vector comprising the nucleic acid of claim 19.21. A cell comprising a vector or vectors of claim
 20. 22. A method forincreasing a subject's fertility, the method comprising administering tothe subject an amount of the fusion protein of any one of claims 1, 3,or 4, effective to enhance fertility of the subject.
 23. The method ofclaim 22, wherein the subject is infertile and desires in vitrofertilization.
 24. The method of claim 22, wherein the subject is ahuman.
 25. The method of claim 22, wherein the fusion protein isadministered intravenously, intramuscularly, subcutaneously, orally,buccally, sublingually, nasally, rectally, vaginally, via an aerosol, orvia a pulmonary route.
 26. A method for treating a subject having adisease state responsive to treatment by FSH, the method comprisingadministering to a subject an effective amount of the fusion protein ofany one of claims 1, 3, or
 4. 27. The method of claim 26, wherein thesubject is a human.
 28. The method of claim 26, wherein the fusionprotein is administered intravenously, intramuscularly, subcutaneously,orally, buccally, sublingually, nasally, rectally, vaginally, via anaerosol, or via a pulmonary route.
 29. A method of increasing half-lifeof heterodimeric FSH by conjugating, directly or indirectly through alinker, each of an alpha subunit and a beta subunit of FSH to at leastan FcRn binding partner wherein the head of αFSH is aligned with thehead of βFSH and the tails of each of the respective FcRn bindingpartners are aligned.